Vpu is an HIV accessory gene that induces degradation of CD4 in the endoplasmic reticulum and enhances viral particle release. This project seeks to define the mechanism by which Vpu enhances viral particle release. Preliminary studies in our laboratory show that human cells are restricted for particle assembly and require Vpu for efficient particle production (restrictive/Vpu-responsive). Other cells, notably simian cells, are permissive for efficient assembly and do not respond to Vpu (permissive/Vpu-unresponsive). In heterokaryons, the restrictive phenotype dominates, suggesting strongly that a cellular inhibitor of assembly is present in restrictive cell types. Experiments in Aim 1 of this proposal will further characterize this cellular inhibitor. The Vpu-responsive cellular factor will be measured in cell lines and in human primary T cells and macrophages, and a survey of simian cells will determine if indeed this is a species-specific factor. In Aim 2, the site of the assembly block in restrictive cells will be determined. Biochemical fractionation of pulse-labeled cells will define the kinetics of Gag trafficking in the presence and absence of Vpu. Quantitative live cell imaging techniques will be employed to identify the subcellular location where Gag accumulates in restrictive cells, and to define the subcellular site of action of Vpu. A novel Gag-Gag interaction assay based upon fluorescence resonance energy transfer (FRET) will allow the identification of intracellular Gag multimers in living cells; the influence of Vpu on these assembly intermediates will be assessed. Experiments in Aim 3 will identify the cellular inhibitor of assembly that is overcome by Vpu. An expression cloning strategy will be utilized to identify cellular factor(s) in restrictive cells that inhibit HIV assembly and are overcome by Vpu. In parallel, a subtractive hybridization approach utilizing restrictive and permissive Jurkat cell clones will identify candidate cellular cDNAs that restrict assembly. These studies will define the mechanism by which Vpu enhances HIV particle release and will characterize a novel host cell restriction to HIV replication that is present in human cells.